AbstractSulfa drugs are an important class of pharmaceuticals in the treatment of bacterial infections. The amido/imido tautomerism of these molecules in their neutral form has been widely discussed in the literature. Here, we study the protonation preferences of sulfa drugs upon electrospray ionization (ESI) using IR action spectroscopy of the ionized gas-phase molecules in a mass spectrometer. Our set of molecules includes sulfanilamide (SA), the progenitor of the family of sulfa drugs, and the actual, sulfonamide nitrogen substituted, sulfa drugs sulfamethoxazole (SMX), sulfisoxazole (SIX), sulfamethizole (SMZ), sulfathiazole (STZ), sulfapyridine (SP) and sulfaguanidine (SG). IR multiple photon dissociation (IRMPD) spectra were recorded for the protonated sulfa drugs using a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR-MS) and an optical parametric oscillator/amplifier (OPO/OPA) as well as the FELIX free electron laser (FEL) as IR sources. The OPO provides tunable IR radiation in the NH stretch region (3100–3700 cm$^{-1}$ - 1 ), while the FEL covers the fingerprint region (520–1750 cm$^{-1}$ - 1 ). Comparison of experimental IR spectra with spectra predicted using density functional theory allowed us to determine the gas-phase protonation site. For SA, the sulfonamide NH$_2$ 2 group was identified as the protonation site, which contrasts the situation in solution, where the anilinic NH$_2$ 2 group is protonated. For the derivative sulfa drugs, the favored protonation site is the nitrogen atom included in the heterocycle, except for SG, where protonation occurs at the sulfonamide nitrogen atom. The theoretical investigations show that the identified protonation isomers correspond to the lowest-energy gas-phase structures.